Process for glucan preparation and therapeutic uses of glucan

ABSTRACT

A process for the production of β-3-(1,3)(1,6) glucan from a glucan containing cellular source is described, together with compositions and uses/methods of treatment involving glucan. The process of the invention comprises the steps of: (a) extracting glucan containing cells with alkali and heat, in order to remove alkali soluble components; (b) acid extracting the cells of step (a) with an acid and heat to form a suspension; (c) extracting the suspension obtained of step (b) or recovered hydrolyzed cells with an organic solvent which is non-miscible with water and which has a density greater than that of water separating the resultant aqueous phase, solvent containing phase and interface so that substantially only the aqueous phase comprising β-(1,3)(1,6) glucan particulate material remains; wherein the extraction with said organic solvent provides separation of glucan subgroups comprising branched β-(1,3)(1,6)-glucan, and essentially unbranched β-(1,3) glucan which is associated with residual non-glucan contaminents; and (d) drying the glucan material from step (c) to give microparticulate glucan.

FIELD OF INVENTION

The present invention relates to a process for the extraction of anaturally occurring carbohydrate (glucan) from microorganisms as well asthe glucan produced by this process. The invention also relates to noveltherapeutic users of glucan.

BACKGROUND OF THE INVENTION

Glucan is a generic term referring to an oligo- or polysaccharidecomposed predominantly or wholly of the monosaccharide D-glucose.Glucans are widely distributed in nature with many thousands of formspossible as a result of the highly variable manner in which theindividual glucose units can be joined (glucosidic linkages) as well asthe overall steric shape of the parent molecule.

The glucan referred to in this invention typically is a linear chain ofmultiple glucopyranose units with a variable number of side-branches ofrelatively short length. The glucosidic linkages are predominantly (notless than 90%) β-1,3 type with a lower number (not greater than 10%) ofβ-1,6 type linkages; the β-1,3 linkages form the bulk of the backbone ofthe molecule, while the β-1,6 linkages occur predominantly in theside-branches. The chemical name of this form of glucan ispoly-(1,3)-β-D-glucopyranosyl-(1,6)-β-D-glucopyranose. Glucan is welldescribed molecule.

The form of glucan is found principally in the cell wall of most fungi(including yeasts and moulds) and in some bacteria. Glucan, incombination with other polysaccharides such as mannan and chitin, isresponsible for the shape and mechanical strength of the cell wall. Theglucan typically accounts for approximately 40% to 50% of the weight ofthe cell wall in these cells.

The chemical structure of fungal cell wall glucan has been studied indetail, with the following sentinel articles being incorporated hereinby reference—Bacon et al (1969); Manners et al (1973).

Fungal cell wall glucans have long been used in industry, particularlythe food industry, usually in a semi-purified form. Their uses haveincluded use as stabilizers, binders, thickeners and surface activematerials.

It also has been known for some forty years that fungal cell wallglucans are biologically active, exerting a number of effects on thereticuloendothelial and immune systems of animals. The outstandingbiological effect in this regard is their ability to stimulate nonspecifically the activity of the body's primary defence cells—themacrophage and the neutrophil. This is thought to be due to receptors toβ-1,3 glucan displayed on the surface of these cells (Czop and Austen.1985). The interaction between glucan and its receptor producing suchstimulatory effects as enhanced phagocytosis (Riggi and Di Luzio, 1961),increased cell size (Patchen and Lotzova, 1980), enhanced cellproliferation (Deimann and Fahimi, 1979), enhanced adherence andchemotactic activity (Niskanen et al, 1978), and production of a widerange of cytokines and leukotrienes (Sherwood et al. 1986, 1987).

The aforementioned biological responses to fungal cell wall glucan havebeen reported to result in a number of clinical effects including:enhanced resistance to infections with fungi (Williams et al. 1978),bacteria (Williams et al, 1983), viruses (Williams and Di Luzio, 1985),protozoa (Cook et al, 1979) following systemic application: enhancedantitumour activity following systemic application (Williams et al.1985) or intralesional application (Mansell et al. 1975); and enhancedimmune responsiveness following systemic application (Maeda and Chihara,1973). It will be readily seen that these clinical effects are highlybeneficial and important and represent an opportunity to develop novelpharmaceutics based on fungal cell wall glucans, such pharmaceuticshaving potentially wide application in both veterinary and humanmedicine.

Of the various fungal cell wall glucans tested, that from the yeastSaccharomyces cerevisiae has proven to be acceptable in terms ofefficacy and safety as an immune stimulant in animals and humans.Hereinafter this will be referred to as Saccharomyces cerevisiae(“Sc”)-glucan. Predominantly or wholly β-1,3 glucans from other fungi,bacteria or plants from the Graminaceae family have been shown to beimmunostimulatory in animals but compared to Sc-glucan either are not aspotent or if they do have comparable or greater potency then that isusually associated with a higher level of undesirable side-effects.

Sc-glucan has been shown to be biologically active as an immunestimulant in animals in various forms. These include (a) a largemolecular weight (typically greater than 3×10⁶ d), water-insoluble,microparticulate form, or (b) smaller molecular weight (typically lessthan 500,000 d) forms which are dispersible or soluble in water.Water-solubility is described as being achieved either through cleavageof the large microparticulate glucan form to smaller molecules usingprocesses such as enzymatic digestion or vigorous pH adjustments, or bycomplexing to salts such as amines, sulphates and phosphates. Theprincipal advantage of the smaller, water-soluble form vs the largermicroparticulate form is that it is safer when given by parenteralroutes of administration such as intravenously. Also, it is likely thatthe smaller sized molecules are more bio-available on a molar basis.

To date it has neither been technically possible nor economicallyfeasible to synthesise glucan on a commercial basis. Thus preparation ofcommercial quantities of β-1,3 glucan for therapeutic uses requires thatit be extracted from fungi, bacteria, algae or cereal grains.

DESCRIPTION OF THE PRIOR ART

A number of different processes are described for the preparation ofSc-glucan for pharmaceutical use. A common feature of these differentprocesses is the extraction of microparticulate glucan as the primarystep; the glucan is either then used in the final therapeuticformulation in that microparticulate form or is further processed to asmaller molecular weight material (“soluble glucan”) by modification ofits chemical and/or spatial structures.

(i) Microparticulate glucan

The extraction of Sc-glucan from whole yeast cells depends on the factthat the bulk of the cell wall glucan is insoluble in water, strongalkali, acid and organic solvents whereas all other cell wall componentsare soluble in one or more of these solutions.

The essential principles of extraction of Sc-glucan are (i) lysis of theyeast cell to allow the intact cell walls to be separated from the lessdense cytoplasmic contents, and (ii) subsequent or concomitantdissolution of unwanted wall components such as other carbohydrates(glycogen, mannan, glucosamine), lipids and proteins using variouscombinations of water, alkali, acid and organic solvents. It ispreferred in such processes that the three-dimensional matrix structureof the cell wall remains unaltered and intact as a cell wall skeleton(also known as a “cell sac”), comprised predominantly ofβ-(1,3)(1,6)-glucan. The cell wall skeletons characteristically arespherical, hollow structures of approximately 4 to 20 u diameter andwith a molecular weight of between approximately 1,000,000 to 3,000,000daltons and they are insoluble in water. This end-product is termedmicroparticulate Sc-glucan.

A number of methods of extraction of microparticulate Sc-glucan areknown, although all are essentially variations of a common method. Thedescribed methods entail the following steps.

1. Contact of whole yeast cells with strong alkali solution (pH 12 to14). This effects lysis of the cells and dissolution of most of thenon-glucan components except lipids. This step is uniformly rigorous inall described processes. The contact usually is repeated two to threetimes using fresh batches of alkali and heat also usually is applied tospeed the reaction time.

2. The cells then are exposed to acid (pH 1 to 5) with heat to effectdissolution of certain residual non-glucan components and to effect somehydrolysis of the glycosidic linkages, principally the β-1,6 linkages inthe side branches and to a minor extent β-1,3 linkages in the glucanbackbone side-branches. The rigour of this step varies considerablybetween the known processes of relatively mild acid treatment where theconformational changes are minimal and many of the side-branches areretained, through to extensive acid treatment where little or noside-branches remain and which permits hydration of the helical glucancoils during subsequent steps to convert to a water-soluble form.

3. Contact of the cell residue with alcohol and heat with or withoutadditional subsequent exposure to solvents, particularly ether orpetroleum ether to effect removal of lipids.

See, for example, Hassid et al (1941), Manners (1973) et al, Di Luzio(1979), and U.S. Pat. Nos. 4,810,694 and 4,992,540.

Prior art methods for the production of microparticulate glucan may beregarded as disadvantageous in one or more respects. These include pooryield (such as less than about 5% w/w), low purity (such as less thanabout 90% purity), extended processing time, significant wasteproduction, and high cost.

(ii) Soluble glucan

Microparticulate Sc-glucan is water insoluble due to the tightly boundtriple helical carbohydrate coils which resist hydration.

There are two principal purposes to seek to solubilize Sc-glucan. Thefirst reason is the risk of microembolization associated with theinjection of microparticulate glucan by intravenous or other parenteralroutes. The second reason is that a reduction in molecular weight of theSc-glucan might reasonably be expected to be associated with increasedbiological efficacy due to greater bioavailability of the glucanmolecules.

Solubilization of microparticulate glucan can be achieved in variousways.

One way is to expose the glucan to a specific enzyme. β-1,3-glucosidasewhich cuts the long linear chain into shorter lengths. The disadvantageof this method is that the enzymic digestion process is difficult tocontrol and can result in excessive hydrolysis of the glucan molecule tomonosaccharides or oligosaccharides which lack immunostimulatoryactivity.

Another way is to attach charged groups such as phosphate (U.S. Pat.Nos. 4,739,046; 4,761,402), sulphate (Williams et al, 1991) and amine(U.S. Pat. No. 4,707,471) which permit hydration of the molecule. Bothphosphorylated (U.S. Pat. No. 4,761,042) and sulphated (Williams et al,1991) Sc-glucans retain their immunostimulatory activity and are highlywater soluble. A disadvantage of these methods is that of an additionalstep of complexity in processing operations, which may add considerablyto overall manufacturing cost.

A third approach to solubilization is by sequential alkali/acid/alkalihydrolysis. This was first demonstrated by Bacon et al (1969) who showedthat microparticulate Sc-glucan extracted in the traditional manner byrepeated NaOH exposures followed by an acid wash, almost completelydissolved when subsequently exposed to 3% NaOH at 75° C. This phenomenonis described again in PCT/U.S. application Ser. No. 90/050,41 wherebymicroparticulate Sc-glucan following exposure to acetic acid or formicacid is exposed to IN NaOH for one to two hours at 80° C. to 100° C. Theresultant glucan is of widely heterogenous molecular weight with a highpolydispersity index associated with the presence of glucan moleculesvarying in size from approximately 5,000 d up to approximately 800,000d. That patent application describes further purification bydiafiltration of the hydrolyzed glucan to isolate glucan molecules ofdefined molecular weight from the heterogenous molecular weight speciesproduced, and the use of various resins to remove contaminatingproteinaceous and lipid components.

The present invention insofar as it is concerned with processes for theproduction of glucan, whether in microparticulate or non-particulateform (“soluble”), seeks to overcome one or more of theproblems/deficiencies of prior art processes for the production ofglucan.

In addition, as described hereinafter, this invention is also concernedwith novel therapeutic uses of glucan, whether produced by the methodherein, or other methods known in the prior art.

SUMMARY OF THE INVENTION

In accordance with a first aspect of this invention there is provided aprocess for production of β-(1,3)(1,6) glucan from a glucan containingcellular source which comprises the steps of:

(a) extracting glucan containing cells with alkali and heat in order toremove alkali soluble components;

(b) acid extracting the cells obtained from step (a) with an acid andheat to form a suspension;

(c) extracting the suspension obtained from step (b) or recoveredhydrolyzed cells with an organic solvent which is non-miscible withwater and which has a density greater than that of water and separatingthe resultant aqueous phase, solvent containing phase and interface sothat substantially only the aqueous phase comprising glucan particulatematerial suspended in water remains;

wherein the extraction with said organic solvent provides separation ofglucan subgroups comprising branched β-(1,3)(1,6)-glucan, andessentially unbranched β-(1,3) glucan which is associated with residualnon-glucan contaminents: and

(d) drying the glucan material from step (c) to give particulate glucan.

In order to produce a soluble glucan, step (d) of the above process isomitted and the pH of the solvent extracted aqueous phase comprisingglucan particulate material is raised from an acidic pH, to a basic pHso as to effect solubilization of the glucan particles. This step iscarried out at a temperature below about 60° C., preferably betweenabout 2° C. to about 25° C. more preferably between about 2° C. to about8° C., for a time sufficient to achieve solubilization of the glucanparticles. Alternatively, soluble glucan may be prepared by suspendingthe particulate glucan of step (d) in an aqueous alkali solution so asto effect solubilization of the glucan particles. Temperate conditionsare set out above.

The pH of the solubilized glucan may then be adjusted as required togive a pharmaceutical product.

In another aspect this invention is directed to the use of glucan forthe manufacture of a medicament for the treatment of skin ulceration orbone fracture or the enhancement of fixation of implanted orthopaedicdevices, or the prevention/treatment of ultraviolet light induced skindamage.

In a further aspect this invention is concerned with a method for thetreatment of skin ulceration or bone fracture or the enhancement offixation of implanted orthopaedic devices, or the prevention/treatmentof ultraviolet light induced skin damage, which comprises administeringto a subject glucan in association with one or more pharmaceutically orveterinarily acceptable carriers or excipients.

In another aspect this invention is concerned with an agent for thetreatment of skin ulceration or bone fracture or the enhancement offixation of implanted orthopaedic devices, or for theprevention/treatment of ultraviolet light induced skin damage whichcomprises glucan optionally in associate with one or morepharmaceutically acceptable carriers or excipients.

DETAILED DESCRIPTION OF THE INTENTION

The process described in detail hereafter sets out the production ofβ-(1,3)(1,6) glucan from a cellular glucan source, which is suitable fora variety of pharmaceutical purposes.

In a first aspect the invention is concerned with a process for theproduction of glucan from a glucan containing cellular source. Thisprocess comprises the steps of:

(a) extracting glucan containing cells with alkali and heat in order toremove alkali soluble components;

(b) acid extracting the cells of step (a) with an acid and heat to forma suspension:

(c) extracting the suspension obtained of step (b) or recoveredhydrolyzed cells with an organic solvent which is non-miscible withwater and which has a density greater than that of water and separatingthe resultant aqueous phase, solvent containing phase and interface sothat substantially only the aqueous phase comprising glucan particulatematerial remains;

wherein the extraction with said organic solvent provides separation ofglucan subgroups comprising branched β-(1,3)(1,6)-glucan, andessentially unbranched β-(1,3) glucan which is associated with residualnon-glucan contaminents: and

(d) drying the glucan material from step (c) to give particulate glucan.

While yeast cells generally and the yeast strain Saccharomycescerevisiae in particular are the preferred source of the glucanaccording to this invention, any other cells such as fungi or bacteriacontaining glucan with the properties described herein may be used. Awide range of other yeast and fungal strains can be used in the presentprocess and the following types are included by way of example:Sclerotium spp, Shizophyllum spp, Pichia spp, Hansenula spp, Candidaspp, Saccharomyces spp, Torulopsis spp.

In the case of Saccharomyces cerevisiae the yeast may be grownspecifically for the purpose of extraction of Sc-glucan or may be from acommercial source such as yeast manufactured for the baking industry orspent yeast from the brewing industry.

The first step according to the process of the present inventioninvolves treatment of the yeast cells with alkali and heat to effectcytolysis and hydrolysis of the cytoplasmic components and predominantcell wall components including mannan, chitin (glucosamine), proteinsand glycogen. This treatment (which may also be referred to asextraction or hydrolysis) releases non-glucan components into theaqueous phase so that they might readily be separated by a process suchas centrifugation from the intact cell walls comprising largely glucan.The extent of non-glucan component removal can be readily assessed bystandard analytical techniques, such as those described in U.S. Pat. No.4,992,540.

The alkali extraction step may be carried out in aqueous hydroxide offrom about 2% to about 6% concentration (w/v), such as between 3% and 4%(w/v). Sodium hydroxide or potassium hydroxide find particularapplication because of their availability and relatively low cost.However, any other strong alkali solution which has suitable solubilitycharacteristics, for example, calcium hydroxide or lithium hydroxide,can be used. The yeast is left in contact with the alkali for a timesufficient to remove alkali soluble non-glucan components. Non-glucancomponents are removed more rapidly at higher temperatures. Thedigestion may be carried out at temperatures of from about 50° C. toabout 120° C., requiring exposure times to the alkali of between fifteenminutes and sixteen hours. During alkali exposure, the process ofcytolysis and dissolution of non-glucan components may be facilitated byvigorous mixing of the yeast suspension using appropriate methods suchas by example a stirring apparatus or an emulsifying pump.

Repeat exposure of the yeast cells to fresh batches of alkali solutionassists in removing non-glucan material, particularly protein, from thedisrupted yeast cells. The number of alkali treatments is not limitingon the invention. However, the process should be repeated until it isapparent that the cells have been lysed and the majority of non-glucanalkali soluble components extracted. This can be confirmed by visual orchemical analysis (such as by gas chromatography/mass spectrometry).Treatments using low strengths of hydroxide solution and lowtemperatures of alkali exposure generally may require increased numbersof separate alkali exposures. By way of example, alkali treatment may berepeated from one to six times.

In one embodiment of the present invention in relation to the alkalidigestion phase, dried commercial Saccharomyces cerevisiae is suspendedto 10% w/v in sodium hydroxide at a strength of between 3% and 4% and attemperatures of between 80° C. and 100° C. It has been found that threealkali treatments are typically required for a high purity product.Following each separate alkali exposure, the disrupted yeast cells andthe supernatant solution are separated by any method which is known tothis art including, for example, filtration, centrifugation orchromatography. These separation techniques are referred to by way ofexample only and are not limiting to the process of the presentinvention.

The next step in the process involves the exposure of thealkali-insoluble cell wall sacs to acid, generally at a pH from about2.0 to 6, preferably between 3.5 to 4.5. This procedure dissolves someresidual contaminants such as mannan and chitin. However, the principalreason for this step is to induce conformational alterations to theglucan molecule. The principal alteration is a reduction in the numberof β-1,6 side-branches (Table 1). In native cell wall Sc-glucan, theproportions of glycosidic linkages is approximately 90% β-1,3 and 10%β-1,6. Acid hydrolysis removes the β-1,6 side-branches with the degreeof hydrolysis 30 being related directly to the vigour of the acidtreatment; strong acid treatment (low pH and high temperature, such aspH less than 2 and temperatures above about 100° C.) can effectivelyremove all side-branches whereas less vigorous treatment will leaveβ-1,6 linkages in the proportions of between approximately 1% and 8%.

TABLE 1 Effect of acid exposure (phosphoric acid, ph 4.5, 100° C. 30minutes) on the chemical composition of alkali insoluble Sc-glucan asmeasured by gas chromatography-mass spectroscopy Pre-acid Post-acidMannan (% w/w monosaccharides) 0.5 0 β-glycosidic linkages (mol %): 1,354.2 94.4 1,4 7.1 0 1,3,4 0.7 0.2 1,2,3 2.2 0.5 1,3,6 5.6 2.2 1,6 9.7 01,4,6 0.8 0 1,2,3,4 1.5 0 1,3,4,6 1.9 0 1,2,3,6 0.4 0 Terminal-glc 6.42.9 glucitol hexaacetate 10.8 0

It is known in the art that the degree of branching of β-1,3-glucanmolecules has an important influence on biological function. Forexample, it is known that highly branched glucans such as lentinaninduce pro-inflammatory effects in addition to immunostimulatory effectsand that the pro-inflammatory effects may be associated with adverseclinical side-effects, unbranched Sc-glucans such as those described inU.S. Pat. Nos. 4,739,046, 4,761,402 and 4,7707,471 or Sc-glucan withreduced branching such as that detailed in PCT/U.S. Pat. No. 90/05,041are known to avoid or to greatly diminish pro-inflammatory effects andtherefore be more desirable therapeutic agents clinically. Hitherto,however, the structure/function relationship in terms ofimmunostimulatory capacity and promotion of tissue repair in particularhas not been defined. The inventors have defined the optimal degree ofbranching by comparing the efficacy of differently branched glucanpreparations in an animal wound healing model. For example, afull-thickness surgical skin incision may be made in experimentalanimals such as laboratory rats. Glucan is applied to the woundimmediately following wounding and the wound then allowed to heal. Sevendays later the degree of healing is tested by determining the amount offorce required to separate the apposing wound edges (referred to as‘wound breaking strength’). The results of this experiment aresummarised in Table 2. It can be seen that where the degree of branchingis measured in terms of the proportion of β-1,3:β-1,6 linkages, both alow proportion (90%:10%) as for native glucan and a high proportion(100%:0%) are less effective In the promotion of dermal wound repairthan moderately-branched (98%:2% or 96%:4%) glucan.

TABLE 2 Tensile strength of rat skin wounds (day +7) followingapplication of micro-particulate Sc-glucans with different ratios ofβ-1,3 to β-1,6 glycosidic linkages. Wound tensile strength (g) Treatmentn β-1,3:β-1,6 linkages mean (SD) No glucan 16 — 202 (37) Glucan  890%:10% 252 (45) Glucan 12 96%:4%  358 (49) Glucan  9 98%:2%  339 (38)Glucan 10 100%:0%  285 (52)

1 m,>of glucan was applied at time of operation in oily base to 5 cmlong full-thickness incisional wound.

The nature of the acid used in the acid exposure step is generallyunimportant. Preferably, the acid is employed to provide a pH of theresultant yeast suspension from about pH 2.0 to about 6.0. morepreferably from about pH 3.5 to about 4.5. Suitable acids includehydrochloric, acetic, formic and phosphoric acids.

The process of acid hydrolysis is aided by heating.

The extent of acid treatment, namely pH, temperature and time depends onthe degree of β-1,6 content sought in the glucan product. In order toproduce a glucan product generally containing from 2% to 4% β-1,6linkages, the pH of the solution is selected to be in the range of about2 to about 6, temperature is generally between about 50° C. and about100° C., and the time of reaction from about fifteen minutes to aboutsixteen hours. The extent of β-1,6 linkages in the hydrolyzed glucan canbe readily determined by standard analytical techniques such as nuclearmagnetic resonance (NMR) analysis.

Following the acid exposure stage, the yeast cells predominantly are inthe form of isolated cell wall sacs.

In prior art methods of Sc-glucan preparation it has been proposed toexpose acid extracted glucan containing cells (cell sacs) with alcohol,petroleum ether or diethyl ether, to selectively dissolve remainingnon-glucan components. In contrast, it has been found by the inventorsthat extracting the acidified glucan containing cells with an organicsolvent which is non-miscible with water, that is, has a density greaterthan 1 g/cm³, is particularly and unexpectedly advantageous.Specifically, a single extraction step with such a solvent provides afine discrimination between glucan and non-glucan components, and allowsready separation of glucan subgroups comprising branched glucancontaining both β-1,3 and β-1,6 linkages (which partitions into theaqueous phase) and which is essentially free of non-glucan components(Table 3), and glucan comprising essentially unbranced β-1,3 linkagesonly and which is associated with residual non-glucan membranecomponents such as chitin and protein (which partitions at the interfacebetween the aqueous and organic phase).

TABLE 3 Effect of chloroform extraction on the chemical composition ofalkali/acid treated Sc-glucan. Chemical composition (% w/v) GlucanMannan Protein Chitin Glycogen Lipids Pre- 85.5 0.5 1.4 2.1 4.3 5.6chloroform treatment Post- 98.5 <0.1 0.3 0.2 0.4 — chloroform treatment

The branched β-(1,3)(1,6) glucan subgroup which partitions into theaqueous phase may contain minor or trace amounts of unbranched β-1,3glucan (less than about 5%, generally less than about 2%, morespecifically less than about 0.5% (w/w)) and trace amounts of non-glucancontaminents. It may thus be regarded as essentially branchedβ-(1,3)(1,6) glucan which is free of other glucan and non-glucancomponents. The unbranched β-(1,3) glucan subgroup which is associatedwith non-glucan contaminents and which partitions into the interfacebetween the aqueous phase and organic phase can be readily removed. Itmay contain very minor or trace amounts of branched β-(1,3)(1,6) glucan(generally less than about 1.3% (w/w)) and hence is considered to beessentially unbranched.

Unbranched β-(1,3) glucan may comprise up to 20% of total glucan content(w/w) following alkali/acid/solvent treatment, the remainder comprisingbranched β-(1,3)(1,6) glucan.

Branched β-(1,3)(1,6) glucan is the most potent biologically active formof glucan in terms of wound healing as shown in Table 4.

TABLE 4 Tensile strength of rat skin wounds (day +7) followingapplication of Sc-glucans recovered from either the aqueous or interfacephase following chloroform extraction. Wound tensile strength (g)Treatment n Post-chloroform phase mean (SD) No glucan 12 — 185 (21)Glucan 14 Aqueous 345 (57) Glucan  8 Interface 267 (59)

Thus it can be readily appreciated, particularly in terms of efficacy ofpromotion of dermal wound healing and the production of pure glucanmolecules, that there is much potential therapeutic benefit inseparating the two glucan sub-groups by chloroform extraction(representative of solvents having a density greater than 1).

Solvents which may be used include chloroform (δ=1.48 g/cm³)methylchloroform (δ=1.33), tetrachloroethane (δ=1.5953 g/cm³),dichloromethane (δ=1.325), and carbon tetrachloride (δ=595 g/cm³).Preferably the solvent is volatile to allow ease of removal of anyresidual. Chloroform is particularly preferred.

For convenience of description the description hereafter will refer tothe use of the preferred solvent, chloroform. The invention is not solimited, and any solvent having the requisite density may be used in theinvention.

The chloroform extraction may be performed in the following manner. Theacidified aqueous suspension containing microparticulate glucan may bereacted directly with chloroform in the approximate ratio ofchloroform:aqueous cell suspension of between 1:10 and 5:1. preferably1:4. The yeast cells may comprise (by volume) between about 1% and about90% of the aqueous suspension, such as between about 30% and 50%. It hasbeen found that the process of extraction with chloroform is notfacilitated by heat and preferably is carried out at room temperature.The chloroform and aqueous phases are mixed vigorously using standardmethods including, for example, stirring apparatuses or an emulsifyingpump so as to effect good contact between the chloroform micelles andthe yeast cells. The duration of mixing is a function of the volume ofthe suspension and the stirring or mixing capacity of the stirring ormixing apparatus. An example by way of illustration is that anemulsifying pump with a pumping capacity of 100 L per minute would berequired to mix a suspension volume of 500 L for about ten minutes.

A notable feature of the chloroform extraction step is that the yeastmaterial changes nature both in colour (converting from a light-graycolour to a white colour) and in form (converting from a material withtypical cellular characteristics (cell sacs) in suspension to aflocculent particulate material). The bleaching and flocculating effectsobserved as a result of contact with chloroform (and other solventshaving the requisite density referred to above), have not been observedwith other organic solvents which have a density less than 1 g/cm³.Solvents which have been tested in this regard include acetone, diethylether, petroleum ether, methylene dichloride, ethyl acetate, ethanol,methanol and butanol. Following chloroform exposure and mixing such asbetween about five and ten minutes, the suspension is allowed to settleand quickly separates into three distinct phases—a lower organic phase,an upper aqueous phase, and an interface between those two phases whichis coloured gray. The three phases are well differentiated and readilyseparated. The organic phase is slightly opaque and contains lipids butno glucan. The aqueous phase contains glucan particles suspended inwater. The interface contains a mixture of glucan, protein, and chitinand lipids. When analyzed by NMR, the glucan in the aqueous phasecontains a mixture of β-1,3 and β-1,6 glycosidic linkages in theapproximate ratio of 95% to 98%:2% to 5% respectively. The glucan in theinterface phase contains predominantly unbranched β-1,3 glycosidiclinkages (generally 98 to 100% β-1.3:0% to 2% β-1,6), Effectiveseparation of branched β-1,3 glucan, unbranched glucan, and non-glucancontaminants is achieved.

This separation of glucan particles based on their level of non-glucancontaminants has been found only with solvents having the densitymentioned above, and not with other commonly available organic solventshaving a density less than 1 g/cm³. Without being bound by anyparticular theory the fine discrimination in separating glucan speciesas exemplified by chloroform, may be due to the combination oflipophilic nature of the solvents and their specific density. This mayallow differential separation by weight of cell wall glucan moleculeswhich are associated with other carbohydrates and non-carbohydrates. Theglucan and non-glucan molecules In this interface phase can be separatedsubsequently by evaporation of the chloroform followed by contact of theresidue with ether and ethanol to effect dissolution of the non-glucancomponent, leaving essentially unbranched β-1,3 glucan.

The aqueous glucan suspension collected following the specific solventexposure step may be boiled briefly to effect complete removal of anyresidual solvent and the glucan particles th en dried by standardmethods including, for example, freeze-drying, heating, air-drying orspray-drying. The final product is a slightly off-white, flocculentpowder comprising particles of Sc-glucan with a diameter typically ofbetween about 1 u up to 10 u with a median diameter of about 3 u (suchparticles may be referred to as microparticulate glucan). The powder maybe milled using standard procedures (hammer milling or ball milling) togive particles of desired size.

The separation of predominanly branched and uncontaminated glucan, fromrelatively unbranched glucan associated with non glucan components, isnot achieved where glucan particles are reacted with alcohol prior toreaction with a solvent have density greater than 1, such as chloroform.This is an unexpected finding.

Prior art description of the use of organic solvents to remove lipidsfrom particulate glucan preparations failed to appreciate thediscriminating effects of solvents having a density greater than 1 inseparating predominantly branched, uncontaminated glucan frompredominantly unbranched contaminated glucan. Th is invention may thusbe regarded as a selection which confers substantial advantage asdiscussed above.

The microparticulate Sc-glucan produced by this process can be used as atherapeutic in this form. Some examples of use are application forrepair of tissues such as skin and bone and bowel where themicroparticulate Sc-glucan is applied in formulations such as a powderor cream or lotion or can be used in wound dressings such as bandages orhydrocolloid dressings. Conventional topical formulations may beutilized as are well known in the art and described hereafter.

The process of the invention described above gives rise to a high purityproduct, having a highly potent bioactivity (as it may comprise glucanhaving only β-1,3 and β-1,6 linkages) which is achieved with shortprocessing time, and high yield. Table 5 demonstrates this by comparingglucan produced according to this invention with glucan preparedaccording to the procedures of Hassid et al (1941), Di Luzio et al(1979), Manners et al (1973), and Jamas (U.S. Pat. No. 4,992,540).

TABLE 5 Comparison of four standard methods of extraction ofmicroparticulate Sc-glucan Processing Glucan Time Yield Component levels(% w/w) Method (days) % Glucan Mannan Glycogen Protein Chitin Hassid etal 8 7.8 91.7 0.4 4.5 2.9 0.4 Di Luzio et al 12  2.0 98.1 0.3 0.5 0.70.2 Manners et al 18  12.1  73,8 2.0 9.8 8.6 5.8 Jamas et al 2 7.4 94.60.3 3.1 0.8 1.1 The present 2 7.7 98.5 <0.1  0.4 0.3 0.2 invention

The process of this invention also provides for the conversion ofparticulate glucan to glucan molecules of smaller molecular weight inthe form of a solution, dispersion or colloid or gel which would besuitable for pharmaceutical, such as parenteral use. Such material mayshow enhanced bioactivity through the greater availability of glucanligands for cytophilic glucan receptors. These glucan preparations maybe regarded as providing glucan in a soluble form, where glucanparticles dissolve in the aqueous phase to give a visually clearsolution, or are otherwise hydrated to the extent that they form adispersion or colloid, or are in the form of a gel. For convenience,these forms may be referred to as soluble glucan.

In the prior art it has been proposed to convert particulate glucan tosoluble glucan using rigorous heat treatement (generally at 75° C. orgreater) in the presence of alkali (Bacon et al 1969). In anotherproposal, the particulate glucan was treated with strong acid (90%formic acid) prior to exposure to alkali and heat. These approachessuffer from a number of disadvantages which include the production ofheterogenous glucan products of wide polydispersity which are unsuitablefor pharmaceutical use without size fractionation, relativeinconvenience, high cost, and production of waste materials.

It has been found by the inventors that the glucan purified as describedabove is readily solubilised in alkali at low temperatures (particularlybetween about 2° C. and about 8° C.). In the present invention, solventextraction of acid treated cell wall sacs with a solvent which has adensity greater than 1, where glucan partitioning takes place withsubsequent separation and isolation of branched glucans, enablessolubilisation in alkali at low temperatures. It is otherwise notpossible to produce soluble glucan having the properties describedhereafter.

In order to produce soluble glucan, step (d) of the process describedabove may be omitted and the pH of the solvent extracted aqueous phasecomprising glucan particulate material may be raised from an acidic pHto a basic pH so as to effect solubilization of the glucan particles.This step is carried out at a temperature below 60° C., preferably fromabout 2° C. to about 25° C., more preferably from about 2° C. to about8° C. for a time sufficient to achieve solubilization of the glucanparticles. Alternatively, soluble glucan may be prepared from glucan ofstep (d) of the above process by reacting the particlate glucan with anaqueous alkali solution so as to effect solubilization of the glucan,particles. Temperature conditions are again below 60° C. as specifiedabove.

An unexpected consequence of the present invention is that after alkalisolubilisation a glucan material having a small polydispersity index(generally less than about 5, more particularly less than about 3)results. This is highly desirable for pharmaceutical agents.Furthermore, no additional size fractionation steps are required. Thisis contrary to prior art teachings as set out above.

In one embodiment, microparticulate glucan isolated as described abovemay be suspended in NaOH solution at a strength of between about 2% and10% (pH between pH 10 and pH 14.5) but preferably 5%; the suspensioncontains between about 0.1 and about 30% (w/w) glucan, such as 5%. Aparticular feature of this reaction step as discussed above, is thatcontrary to the known art it does not require prior exposure to strongacid or applied heat or vigorous agitation; the reaction is found tooccur most advantageously at low temperatures (preferably between 2° C.to 8° C.) and with little or no mixing; the reaction time is generallybetween about one and twenty four hours, such as two hours. Betweenabout 90% to 99% of the glucan particles are converted (through alkalinehydrolysis) to suspended small molecular weight molecules over thereaction time. At the conclusion of the reaction the undissolvedparticles are removed by standard methods such as, for example,centrifugation or filtration and the pH of the suspension adjusted theaddition of HCl (say from pH 8 to pH 10). This soluble glucan may beused as a pharmaceutical product. The glucan solution may then beadjusted to isotonicity by standard methods such as dialysis orultrafiltration.

The glucan material produced by this method has a molecular weight rangebetween approximately 60,000 to 250,000 with a mean of about 140,000daltons, with a mean polydispersity index of about 2.4. Betweenapproximately 70% and 85% of the glucan molecules are within 15% of themean molecular weight and it is found that this result is highlyreproducible with different batches. This low polydispersity indexindicates relatively high homogeneity. It is thus entirely suitable foruse as a pharmaceutical. It is found that this material has highbiological potency, as measured, for example, in the promotion of tissuerepair. In a rat dermal wound repair model, this material isapproximately five times as efficacious as microparticulate Sc-glucanwhen compared on an equivalent molar basis (Table 6).

TABLE 6 Tensile strength of rat skin wounds (day +7) followingapplication of a single topical dose of 1 mg micro-particulate vssoluble Sc-glucan with 96% (β-1,3) and 4% (β-1,6) linkages. Woundtensile strength (g) Treatment n mean (SD) No glucan 12 196 (23)Micro-particulate glucan 14 356 (47) Soluble glucan  8 432 (69)

In that experiment the glucans were administered in a lipophilic creambase, but it would be anticipated that this material could be used as atopical therapeutic in a variety of formulations or could be injected asa parenteral therapeutic.

In a strongly alkaline solution, the soluble glucan molecules occurprincipally as triple helices but with little or no polymerisation ofindependent helical structures. The effect of lowering the pH of theglucan solution is to predispose the glucan molecules to polymerisationleading to gel formation. At a pH below approximately 9.0 there isprogressive polymerisation of adjacent helical structures. It isobserved that the degree of polymerisation of the glucan molecules isrelated directly to the concentration of the glucan solution. Where theglucan solution is to be diluted and dispersed in a carrier vehicle andit is desirable to minimise the degree of polymerisation, theconcentration of the glucan solution is generally less than 10 mg/mL,and preferably no greater than 5 mg/mL prior to adjustement of the pHfrom a strongly alkaline state (around pH 13). In other instances it maybe desirable to have the final glucan solution as a gel and this isachieved if the concentration of the glucan solution prior to pHadjustment is greater than 10 mg/mL (10% w/w) and preferably greaterthan 15 mg/mL (15% w/w), for example up to about 30% w/w. It is foundthat this gel state is a convenient form for topical application,requiring little or no additional formulation.

It can be seen that the present manufacturing process represents asignificant advance over the current state of the art in this field.Compared to other known manufacturing processes, the present processyields an end-product which has greater purity, is manufactured in ashorter time, has greater efficiency of yield, produces a glucanmolecule of distinctive chemical structure, and produces a product ofdesired homogeneity without the necessity of elaborate and expensiveseparation techniques.

It readily would be appreciated that these advantages lead toconsiderable cost savings, with the availability of a less expensivematerial thus allowing wider application of Sc-glucan as a therapeuticin both veterinary and human medicine than is currently available.

The applications for which the microparticulate Sc-glucan produced bythe process of the present invention are suitable include thoseapplications in particular where the risk of direct entry of thematerial to the bloodstream is minimal and these include by way ofexample oral application, topical application, intradermal injection,intramuscular injection, subcutaneous injection, intraperitonealinjection, intrathecal injection, intralesional injection, intratendoninjection, intraligament injection, intraarticular injection, andapplication to fracture sites of bones and cartilage. The therapeuticpurposes include by way of example (a) enhancement of wound repairprocesses in the aforementioned tissues, (b) enhancement of resistanceto infection from bacterial, fungal, viral and protozoal organisms inthe aforementioned tissues, and (c) enhanced local immune responsivenessto carcinogenesis.

The applications for which the small molecular weight Sc-glucan producedby the process of the present invention are suitable include by way ofexample although not being limited to those listed above formicroparticulate Sc-glucan; indeed in these situations the use ofsoluble Sc-glucan may be preferred to that of microparticulate Sc-glucanbecause of various practical considerations such as ease ofadministration or the benefit of administration in a liquid form orbecause of the greater bioavailability of this form. However, smallmolecular weight Sc-glucan has particular indication for thosesituations where penetration of intact tissues (such as trans-epidermalpenetration of intact skin ) is desired or where entry of the materialto the bloodstream may occur inadvertently.

The Sc-glucans produced by the processes of the present invention can bepresented in formulations commonly used in the pharmaceutical andcosmetic industries including, for example ointments, gels, suspension,emulsions, creams, lotions, powders and aqueous solutions. Glucan may beformulated with one or more carriers or excipients as are well known inthe pharmaceutical art (see, for example, Remingtons PharmaceuticalSciences, 17th Edition, Mack Publishing Company, Easton Pa., Ed Osol. etal, which is incorporated herein by way of reference).

Examples of carriers and excipient substances are organic or inorganicsubstances which are suitable for enteral (for example, oral or rectal),parenteral (for example, intravenous injection) or local (for example,topical, dermal, ophthalmic or nasal) administration and which do notreact with the glucan, for example, water or aqueous isotonic salinesolution, lower alcohols, vegetable oils, benzyl alcohols, polyethyleneglycols, glycerol triacetate and other fatty acid glycerides, gelatin,soya lecithin, carbohydrates such as lactose or starch, magnesiumstearate, talc, cellulose and vaseline.

Formulations may include one ore more preservatives, stabilizers and/orwetting agents, emulsifiers, salts for influencing osmotic pressure,buffer substances, colourants, flavourings and/or perfumes.

Glucan may be formulated into sustained release matrices which liberateglucan over time providing what may be regarded as a depot effect.Glucan in the form of a gel, as produced according to an embodiment ofthe aforementioned process, may be directly used as a topicalpharmaceutical product or formulated with appropriate carriers and/orexcipients.

In a further embodiment, this invention is directed to a glucancomposition which consists essentially of branched β-(1,3)(1,6)-glucan,and which is free or essentially free of unbranched β-(1,3) glucan andnon-glucan components. Reference to “essentially free” is to beunderstood to refer to less than about 2% unbranched β-(1,3) glucan,more specifically less than about 0.5% unbranched β(1,3) glucan.

These glucan formulations may comprise glucan in microparticulate form,soluble form or as a gel, optionally formulated or in association withone or more pharmaceutically acceptable carrier or excipients as hereindescribed.

Glucan containing formulations or compositions for therapeutic purposesmay contain from about 0.01% to about 30% (w/w), such as from about 0.1%to about 5%, more particularly from about 0.2% to about 1%, even moreparticularly from about 0.25% to about 0.5% (w/w). These amounts may beregarded as therapeutically effective amounts.

It has surprisingly been found by the inventors that Sc-glucan, whetherproduced according to this invention or by prior art processes may beused in a range of hitherto unsuspected and undescribed therapeuticapplications. These applications include the treatment of ulceration orbone fracture, or the prevention/treatment of ultraviolet light inducedskin damage.

In a further aspect this invention is directed to the use of glucan forthe manufacture of a medicament for the treatment of skin ulceration orbone fracture, or the implantation/fixation of orthopaedic devices, orprevention/treatment of ultraviolet light induced skin damage.

In a further aspect this invention is concerned with the method for thetreatment of skin ulceration or bone fracture, or theimplanation/fixation of orthopaedic devices, or prevention/treatment ofultraviolet light induced skin damage, which comprises administering toa subject glucan in association with one or more pharmaceutically orveterinarily acceptable carriers or excipients.

In a still further aspect of this invention, there is provided an agentfor the treatment of dermal skin ulceration, the enhancement of repairof bone and connective tissue, or the implanation/fixation oforthopaedic devices, or the prevention/treatment of ultraviolet lightinduced skin damage, which agent comprises glucan in association withone or more pharmaceutically or veterinarily acceptable carriers orexcipients.

In these novel therapeutic uses of glucan, an effective amount of glucanis utilised. What constitutes an effective amount will depend on theparticular condition being treated, mode of and form of administration,and like factors. Generally, a composition or medicament will containglucan in an amount from about 0.05% (w/w) to about 30% (w/w), such as0.1 to 5% (w/w), more particularly from about 0.3% to about 1% (w/w),even more particularly from about 0.25% to about 0.5% (w/w).

A particularly advantageous therapeutic application for glucan (such asmicroparticulate, soluble or gel forms manufactured by any of theaforementioned methods, or produced by prior art methods) according tothe present invention is in the treatment of dermal ulceration. It isknown that β-1,3-glucan will promote healing in full-thickness,surgically-created skin wounds in animals and humans with nodysfunctional healing. That is, the topically- or parenterally-appliedglucan is able to accelerate the healing response in superficial woundswith normal healing mechanisms. It generally is assumed that glucanachieves this through activation of wound macrophages. Macrophapes arecritical cells in the healing process, producing a range of cytokinesand growth factors which initiate the various components of the healingcascade—viz, fibroplasia, collagen production, angiogenesis,epithelialisation and collagen cross-linking. The macrophage plays a keymodulatory role in this process, both initiating the process and helpingto ensure that the process proceeds in a coordinated and integratedmanner. It is assumed that a primary effect of the glucan is to producea temporal acceleration of the healing cascade.

Dermal ulcers typically are chronic wounds which have a quite differentset of physiological properties operating within the wound, compared toacute surgical wounds. Whereas the physiology of the healing process iswell described for acute surgical wounds, it is ill defined for chroniculcers. Ulcers typically show poor to negligible healing because ofeither constant irritation or pressure (such as decubitus ulcers orpressure sores) or restricted blood supply (such as in individuals witharterial ischaemia or venous thrombosis) or infection (such as‘tropical’ ulcers) or nerve damage (‘neurotrophic’ ulcers) or diabetes.Ulcers have varying pathologies, and the underlying causes, where known,may be quite distinct. Various types of ulcers which may be treatedaccording to this invention include those associated with physicaltrauma (radiation, thermal burns, decubitus, insect bites), impairedblood flow (arterial, venous), infection (bone, pyogenic, synergisticgangrene, syphilis, tuberculosis, tropical diseases, fungal diseases),neoplasia (primary skin tumour, metastases, leukemia) and neurotrophiclesions, (spinal cord lesions, peripheral neuropathies).

Ulcers associated with dysfunctional healing vary greatly in severity,from superficial wounds extending into the dermis and having a surfacearea of approximately 1-2 cm² up to wounds extending through dermis,subcutaneous tissue and muscle and forming depressions and cavities withvolumes of approximately 500 cm³. The larger ulcers in particular can bedebilitating and restrictive and require intensive and expensive therapyto manage. Control of wound sepsis, regular wound debridement, regulardressings, hypostatic drainage and corrective surgery are just some ofthe standard current therapies. However, currently available‘best-practice’ wound management therapies are not uniformly successful,take considerable lengths of time to produce beneficial results, areassociated with poor rates of patient compliance, generally areexpensive, and are associated with a high incidence of ulcer recurrence.It has been noted by Margolis (J. Dermatological Surgery (1995) 21(2)145-148) that: “a paucity of data exists that adequately addresses theefficacy of any topical agent for the treatment of pressure ulcers”.

It can be seen therefore that in view of the high incidence of ulcers inthe community and the cost to the community of treatment, there is anurgent need to develop improved therapies. Ideally, such a therapyshould have a high rate of success, be convenient to use and produce aclinic response quickly in order to facilitate patient compliance, andpreferably be inexpensive.

A particular difficulty in devising a uniformly successful therapy whichmay be an improvement on current treatment modalities is thenon-unifomity of the different types of ulcers where both the underlyingaetiologic disease processes and the pathophysiology within the woundsshow considerable variation. Confounding this variability, is thegeneral poor understanding of which of the different components of thehealing response is dysfunctional and therefore contributing the primarypathology of the dysfunctional healing response. So that, successfulantagonism of dysfunction of any particular part of the healing cascadein one ulcer type may not necessarily be successful in another ulcertype. In particular, there is no evidence that local wound immunesuppression or macrophage dysfunction are key pathological features orthat enhancement of local immune mechanisms within such ulcers wouldresult in enhanced healing responses as is seen in uncomplicatedsurgical skin wounds with no dysfunctional healing responses.

Thus it was entirely unexpected to find that topical application ofglucan to decubitus, venous stasis and arterial ischaemic ulcers inducedrapid and potent healing responses in those wounds. This was unexpected(a) because the primary causative factor of these ulcer types isimpaired blood supply and there is no evidence to suggest that thiswould be responsive to antagonism by an immune stimulant, and (b)because even where it might be possible to promote the healing response,the impaired vasculature to the wound could be expected to impede thehealing response as is observed with current treatment modalities. Thebeneficial effect of glucan in these ulcer types is even more remarkablegiven that a complete healing response can be achieved in the absence ofother supportive therapies such as sepsis control, hypostatic drainageand correction of the primary cause.

The treatment of decubitus ulcers and venostasis ulcers are particularlypreferred according to this invention, although the invention is notlimited to the treatment of these ulcer conditions.

Decubitus ulcers arise through multiple mechanisms. They are adisastrous complication of immobilization. They may result from shearingforces on the skin, blunt injury to the skin, drugs and prolongedpressure which robs tissue of its blood supply. Irritative orcontaminated injections and prolonged contact with moisture, urine andfaeces also play a prominent role. Diminished blood circulation of theskin is also a substantial risk factor. The ulcers vary in depth andoften extend from skin to a bony pressure point. Treatment is difficultand usually prolonged. Surgical techniques are at present the mostimportant means of achieving optimal healing.

Approximately half of venous ulcers are associated with incompetentperforating veins in the region of the ankle, and constitute a long termproblem in many immobile patients. Ulceration is rarely a manifestationof primary varicose veins but is virtually always associated withincompetence of the popliteal venous valve. Venostasis ulcers are mostoften just proximal or distal to the medial malleolus (bony ankle joint)and often develop at sites of minor trauma or skin infections. Scarringand secondary infection all impair healing and make recurrences commonif healing does occur. The natural history of venous ulceration iscyclic healing and recurrence.

In the case of decubitus ulcers, the glucan preferentially is applied inthe form of a powder (microparticulate glucan) or in a cream or ointmentbase (microparticulate, soluble or gel forms of glucan). Application isgenerally daily and may continue for a time period sufficient for ulcerhealing, such as seven to twenty eight days. It is observed that theresponse to the glucan therapy is apparent clinically within 2-3 dayswith evidence of fresh granulation and epithelial growth. The length oftime required to heal ulcers will vary according to a number of factorssuch as ulcer size, degree of wound sepsis and host nutritional state.Typicallv wound volume is reduced by 50% within 2-3 weeks with completeor near-complete wound closure effected by 4-6 weeks after commencementof glucan therapy. It is noteworthy that most of the decubitus ulcers inwhich glucan effects a healing response have been refractory to standardtherapy including a wide range of topical preparations and wounddressings for periods up to 7 years.

In a similar manner, application of microparticulate, soluble or gelforms of glucan to venostasis and arterial ischaemic ulcers promotesulcer healing. As with the decubitus ulcers, treatment of these ulcerswith glucan leads to a clinical response in the wound within 2-3 daysfollowing the start of glucan therapy with such evidence of healing asthe appearance of fresh granulation tissue and less detritus leading toa cleaner appearance in the wound. Glucan in the form of a powder,cream, lotion, ointment or gel may be topically applied to the ulcersite daily until healing occurs. The chronic nature of the underlyingvascular disorder in these cases means that the predisposition to formsuch ulcers remains with the patient. It may be necessary therefore tocontinue glucan therapy on a long term basis to prevent recurrence.

It can be seen therefore that it is an entirely unexpected observationthat glucan is able to promote the healing processes in skin ulcerswhere the individual components of the healing process are essentiallynormal but are unable to antagonize the dysfunctional cause such asinadequate blood supply, inadequate venous drainage, excessive tissueoedema, infection, constant pressure or other diverse causes.

It is observed that application of glucan to ulcers as described aboveproduces a high rate of therapeutic response. Skin ulcers which eitherare unresponsive or poorly responsive to conventional wound managementpractice, typically respond within several days to treatment with glucanleading in a high proportion of cases to complete healing within severalweeks of treatment. It is found that the glucan is effective in thetreatment of ulcers when applied locally to the wound in various formssuch as a powder, gel, cream, or dressing such as a gauze bandage orcolloidal material, or any other composition generally known to thoseskilled in the art of pharmaceutical formulation.

In a related aspect the treatment of ulcers which respond toconventional therapies (such as normal dressings and ointments) may beaccelerated with glucan administration.

Another unsuspected therapeutic application for glucan (such as,microparticulate, soluble or gel forms manufactured by any of theaforementioned methods, or other processes known in the art) accordingto the present invention is in the treatment of bone fracture. Therepair of fractured bone characteristically is accomplished by a repairprocess which basically is in common with that observed in soft tissuessuch as skin but which differs in some important aspects. In bone, animportant early step in the repair process is the formation of a fibrousstructure known as a callus which bridges the fractured site providing aframework for the repair process and assuring a degree of immobilizationof the fracture site. In due course the callus becomes mineralized,providing continuity with the uninjured bone and undergoes a degree ofremodelling to approximate the original shape of the bone. According tothis aspect of the invention the application of glucan to the site ofinjure enhances the rate of repair of injured bone thus facilitatingfracture treatment. It is observed that the effect of such treatment isearlier induction of the callus formation and earlier organization ofthe connective tissue within that callus to provide a strong fibrousmatrix. The result of this is the establishment of an immobilizingcallus at an earlier time with the important clinical effect of reducingthe risk of dissociation of the fractured edges of the bone. This is ahighly desirable effect in both animals and humans because anydisruption to the fracture site can predispose to delayed healing.Disruption at the fracture site remains a problem, even where methods ofphysical immobilization through such mechanical means as rigid splints(such as casts, bandages, etc.), or implants (such as pins, screws, etc)are used. While it is found that the process of mineralization is notappreciably enhanced by the glucan treatment, it is found that theeffect of glucan in accelerating the callus phase has the effect ofreducing the overall time to complete mineralization.

The glucan preferably is applied directly to the site of bone injury ina form which will maximize the retention of the glucan at the site ofthe fracture. Slow release formulations are well known in the art andare preferably used in these applications. It is found that the viscousgel formed by the embodiment disclosed in this invention whereby ahighly alkaline soluble glucan solution at a concentration of greaterthan 15 mg/mL (from about 15 mg/ml to about 500 mg/ml, more preferablyfrom about 15 mg/ml to about 30 mg/ml) is adjusted to pH 7.5 (Example 4)is a preferred form. This form is sufficiently viscous and non-misciblewith blood and tissue fluids to remain at the site of application forperiods up to 48 hours. An additional advantage of this gel form is thatit is sufficiently tractable to be able to be injected through finegauge needles. In this form, the glucan can be administered by injectionto fracture sites where the fracture is reduced without the need forsurgical exposure of the bone. Alternatively, the gel can beadministered to the fracture site during open surgical reduction offractures.

The potential usefulness of glucan treatment for human bone fractureshas been evidenced in an animal model by the inventors. The rat is astandard model used in experimental medicine for bone fracture researchand generally is regarded as directly predictive of human therapy (Baket. al. 1992). In this animal model the inventors have established thatinjection of 2 mL of 15 mg/mL soluble glucan in a gel form at the siteof a fractured femur resulted in accelerated healing when compared withnon-treated fractures as evidenced by increased tensile strength of thepartially healed bones at 12 days (Example 10).

It can thus be readily envisaged that glucan, being non-toxic andphysiologically acceptable, may find wide application in fracturetreatment in human and animal medicine. For example, a single bolusinjection or application of glucan at the site of fracture will promotehealing and increase tensile strength of the healed bone.

A further unexpected therapeutic benefit is that glucan enhances thefixture of devices such as pins, screws, artificial joints andprostheses fixed or implanted into or onto bone. It is observed that theapplication of glucan (such as by local application of a powder or gel,or by injection) at the site of fixation of the device enhancessignificantly the local inflammatory process which occurs in response tothe contact of the device with bone and generally is an integral part ofthe strength of the bond between the bone and the device.

A particular therapeutic indication for glucan (either microparticulateor soluble forms manufactured by any of the aforementioned methods or byprior art methods) according to the present invention is in thetreatment of injured connective tissues such as tendons and ligamentswhich has not previously been described or suggested. Such tissues aretypically densely fibrous because they are subjected to relatively highstress loads. These injuries include by way of example but are notlimited to (a) acute or chronic inflammation associated with over use orstrain or trauma, such conditions typically being associated withsporting injuries or the syndrome known as Repetitive Strain Injury orexcessive or abnormal stress, and (b) surgery, in particular where thetissue is dissected or transected. It is known that injuries of thiskind in such tissues typically are slow to heal, due in part to therelative difficulty of totally resting the injured tissue because oftheir load bearing functions, but due largely to the characteristicallylower level of activity of all aspects of the tissue healing cascadecompared to that which is seen in soft tissues. An important cause ofthis comparatively lower level of tissue repair activity in tendons andligaments is a more limited blood supply compared to most soft tissues.It is found that application of glucan to the injured tendon or ligamenteither at the time of acute injury such as following surgery or externaltrauma, or with chronic injury such as chronic inflammation will promoteboth the rate of onset and the level of the healing response in thesetissues, leading in the case of surgery to earlier return of normalstrength and function and in the case of inflammation to earlierresolution of the inflammatory process. The glucan may be directlyinjected into the injured tendon or ligament. Although it has beendescribed that glucan is a potent enhancer of wound repair in dermaltissue in healthy tissues, it is not apparent from that knowledge thatglucan has the ability to effect enhanced resolution of chronicinflammatory processes or of enhancing repair processes in tissues withlimited blood supply or where the normal rate of repair is known to berelatively slow.

A further unsuspected therapeutic indication of glucan is theprevention/treatment of ultraviolet light-induced skin damage whichresults from exposure to the sun.

It is well described that ultraviolet light exposure causes damage toskin, particularly long term exposure to sunlight. This is particularlythe case with Caucasians who have light skin colouration whichpredisposes them to photo-ageing and development of certain types ofskin cancers. Both of these problems are prominent within most Westerncommunities.

The detrimental effects of sunlight are due primarily to its ultravioletlight spectrum (UV-A and UV-B). UV-B acts principally within theepidermis and rarely penetrates deeper than the uppermost layers of thedermis, while the longer wave-length UV-A penetrates through the dermallayers. The major detrimental effect of ultraviolet light is damage toproteins, particularly DNA and RNA where it results in dimer formation.Most of these dimers are repaired within several hours although a smallnumber are either not repaired or are mis-repaired and the accumulationof these mis-repairs over a lifetime is thought to be a majorcontributing factor to the development of skin carcinogenesis inchronically sun-exposed individuals.

The two principal outcomes of this damage to proteins in the skin isacute cell damage and mutagenicity. Cell damage is evidenced clinicallyin the acute phase by the symptoms referred to generally as ‘sun-burn’which include ervthema (reddening) and oedema and in the long-term phaseby symptoms referred to generally as ‘photo-ageing’ which include skinthickening and wrinkling; mutagenicity is evidenced by skin cancerdevelopment. A further effect of ultraviolet light which is notclinically apparent is immune depression. Skin has a rich network ofimmune cells that are equally sensitive to the detrimental effects ofultraviolet light as are other skin cells and exposure to ultravioletlight leads to temporary dysfunction of these cells. This dysfunction isrepaired generally within 2-3 days but in this period the skin showsreduced immune capacity such as antigen-presentation. With repeatedultraviolet light exposure such as might be expected in individuals witha lifetime exposure to sunlight, the sun-exposed skin has chronicallyreduced immune function. It is likely that this predisposes to thedevelopment of skin cancer through reduced immune surveillance capacitywithin skin. However, the relative contributions that each of thedifferent effects of ultraviolet light (viz, immune depression, chronicdermal and epidermal cell injury, mutagenicity) has in skin cancerdevelopment and photo-ageing remains unknown.

It has been found surprisingly by the inventors that glucan appliedtopically to skin either following or concominant with ultraviolet lightleads to substantial protection of the skin from ultravioletlight-induced skin damage.

This has been found in experiments conducted with a standard, hairlessmouse strain used as a model to study solar damage to human skin (see,for example. Canfield et al 1985). In this model the mice are exposeddaily for 10 weeks to a minimal erythemal dose of mixed ultravioletlight which simulates the toxic effects of sunlight on skin. Each dailyexposure of ultraviolet light induces a mild erythema and oedema lastingup to about 24 hours and which mimics in appearance a mild ‘sun-burn’ inhumans. With continued irradiation treatment, this on-going damage isreflected in progressive thickening of the skin which histologicallymimics the hyperkeratinisation and elastosis associated withphoto-ageing in chronically sun-expose skin in humans. Pre-malignanttumours begin to appear within several weeks of completion of theultraviolet light treatment regime. Over the ensuing 6-12 months thereis progressive development of pre-malignant and malignant tumours, thehistology and behaviour of which closely mimic the actinic keratoses andpre-malignant and non-melanona skin cancers that develop in humans inresponse to sunlight.

The inventors have found that soluble glucan applied to the skin dailyimmediately following ultraviolet irradiation provides significantprotection from both the acute toxic effects (evidenced by discerniblylesser skin erythema on each morning following the previous day'sirradiation) and the chronic photo-ageing effects (evidenced bysignificantly thinner skin). This effect is particularly unexpectedgiven that β-1,3-glucan is not previously known to protect tissues fromdirect cytotoxic damage and that there is no existing data that eitherconfirms or suggests that β-1,3-glucan antagonises the cytochemical andhistopathological lesions that are consequent to acute or chronicultraviolet irradiation. The ability of glucan in this model toantagonise the acute toxic and chronic photo-ageing effects ofultraviolet irradiation offers a novel and important means of protectionof human skin from the damaging effects of sunlight.

It also has been found by the inventors that soluble glucan appliedtopically to human skin immediately following exposure to sunlightaffords protection from the acute erythemal effects of the ultravioletlight.

It further is found in the hairless mouse model that the glucan affordsconsiderable protection from the development of skin cancers (see FIG. 1hereafter). The majority of tumours at this early stage are benignsessile-based papillomas, as expected; transformation of a proportion ofthese to more malignant intermediate forms culminating in squamous cellcarcinomas is anticipated at a later stage.

Accordingly, glucan may find wide applications in ameliorating theeffects of sunlight in the human population. In this regard, thebeneficial effect of glucan is obtained if it is applied either priorto, during or following sunlight exposure. To this end, it may beformulated into sunscreen formulations or into after-sun or in generalcosmetic formulations such as lotions, creams and gels. The particularbenefits to be gained from the use of Sc-glucan include the following:(a) amelioration of the acute toxic effects of sunlight on skin (‘acutesunburn’); (b) amelioration of the chronic effects of sunlight on skinwhich collectively are known an photo-ageing and include symptoms suchas hyperkeratinisation, skin thickening, elastosis and wrinkling; (c)amelioration of the development of sunlight-induced skin carcinogenesis.

It is to be understood that the novel therapeutic uses for glucan hereindescribed are not limited to glucan produced by the processes describedherein, although this material is preferred. Any prior glucan materialsuch as those described by Hassid et al, Di Luzio et al, Manners et aland Jamas et al (U.S. Pat. Nos. 5,028,703, 5,250,436, 5,082,936 and4,992,540) may be used. Preferably the glucan is Sc-glucan.

This invention will now be described with reference to the followingnon-limiting examples which illustrate various embodiments of theinvention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts dorsal skin fold thickness measurements in mice subjectto U.V. irradiation over 6 weeks, which mice are treated with glucan(-□-) or treated with a non-glucan base lotion (-⋄-).

EXAMPLE 1

Microparticulate glucan is prepared as follows:

A 400 g sample of commercial Saccharomyces cerevisiae in dry form isadded to four liters of 4% w/v sodium hydroxide and heated to 100° C.for one hour with vigorous stirring. The suspension is allowed to coolto between 45° C. and 50° C. before the lysed yeast cells are separatedfrom the alkaline hydrolysate by centrifugation at 800 g for tenminutes. The lysed yeast cells are resuspended in a fresh batch of threeliters of 3% w/v sodium hydroxide and boiled for 15 minutes. Followingseparation by centrifugation, the lysed yeast cells are resuspended in afresh batch of two liters of 3% w/v sodium hydroxide and boiled for 15minutes followed by standing at 70° C. for 16 hours. Followingseparation by centrifugation, the lysed yeast cells are resuspended inwater and boiled for 10 minutes. The latter step is repeated once.Following centrifugation, the lysed yeast cells are resusended in afresh aliquot of 2 L water, the pH adjusted to 4.5 by the addition ofphosphoric acid and the suspension then boiled for thirty minutes. Fivehundred mL of chloroform then is added and the suspension subjected tovigorous agitation for ten minutes, following which the suspension isallowed to settle for 10 minutes in a separating funnel. The suspensionseparates into three distinct phases, a lower organic phase, an upperaqueous phase, and an interface between these two phases which is greycoloured. The lower chloroform phase plus a greyish intermediate phaseare discarded, leaving an aqueous phase which is collected and exposedas before to a fresh batch of 500 mL of chloroform. The final aqueousphase is collected and boiled for 10 minutes to remove any residualchloroform and then dried using a spray-drier.

Analysis of the aqueous phase showed that it contained only branchedβ-(1,3)(1,6) glucan in the ratio of 95 to 98% β-1,3:2 to 5% β-1,6linkages. The organic phase is slightly opaque and contains lipids butno glucan. The intemediate phase (interface) contains unbranched β-(1,3)glucan (98 to 100% β-1,3:0 to 2% β-1,6) associated within chitin,protein and other non-glucan contaminents. Biological tests showed thatthe branched glucan was significantly more biologically active thanunbranched β(1,3) glucan in a wound healing test.

The chemical composition of glucan produced according to this inventionis set forth in Table 7.

TABLE 7 Chemical composition of Sc-glucan produced by the process of thepresent invention. % (by weight) Glucose¹ >98 Mannan¹ <0.2 Protein² <0.5Glycogen³ <0.5 Chitin¹ <0.3 Lipid⁴ not detectable Glycosidic linkages:⁴β-1,3 96-97 β-1,6 3-4 Methods of analysis: ¹= HPLC; ²= Lowry method; ³=GC-MS; ⁴= NMR.

It is clear from this analysis that the end-product is a branchedβ(1,3)(1,6) glucan that is substantially pure, containing only traceamounts of impurities, and containing about 2 to 3% β-1,6 linkages.

EXAMPLE 2

Microparticulate Sc-glucan is prepared as follows:

A 400 g sample of commercial Saccharomyces cerevisiae in dry form isadded to four liters of 4% w/w sodium hydroxide and heated to 100° C.for one hour with vigorous stirring. The suspension is allowed to coolto between 45° C. and 50° C. before the lysed yeast cells are separatedfrom the alkaline hydrolysate by centrifugation at 800 g for tenminutes. The lysed yeast cells are resuspended in a fresh batch of threeliters of 3% w/v sodium hydroxide and boiled for 15 minutes. Followingseparation by centrifugation, the lysed yeast cells are resuspended in afresh batch of two liters of 3% w/v sodium hydroxide and boiled for 15minutes followed by standing at 70° C. for 16 hours. Followingseparation by centrifugation, the lysed yeast cells are resuspended inwater and boiled for 10 minutes. The latter step is repeated once.Following centrifugation, the lysed yeast cells are resusended in afresh aliquot of 2 L water, the pH adjusted to 4.5 by the addition ofhydrochloric acid and the suspension then boiled for ten minutes. Fivehundred mL of chloroform then is added and the suspension subjected tovigorous agitation for ten minutes, following which the suspension isallowed to settle for 10 minutes in a separating funnel. The lowerchloroform phase plus a greyish intermediate phase are discarded,leaving an aqueous phase which is collected and exposed as before to afresh batch of 500 mL of chloroform. The final aqueous phase iscollected and boiled for 10 minutes to remove any residual chloroformand then dried using a spray-drier.

The chemical composition of glucan produced according to this inventionis set forth in Table 8.

TABLE 8 Chemical composition of Sc-glucan produced by the process of thepresent invention. % (by weight) Glucose¹ >98 Mannan¹ <0.2 Protein² <0.5Glycogen³ <0.5 Chitin¹ <0.3 Lipid⁴ not detectable Glycosidic linkages:⁴β-1,3 98-98 β-1,6 1-2 Methods of analysis: ¹= HPLC; ²= Lowry method; ³=GC-MS; ⁴= NMR.

It can be seen that compared to the end-product material obtained inExample 1, this material has has a similar degree of purity but hasslightly fewer β-1,6-glucan linkages indicating a lesser degree ofside-branching.

EXAMPLE 3

A protocol for the preparation of minimally-polymerised, solubleSc-glucan according to the present invention is as follows.

Microparticulate Sc-glucan is produced as detailed in Example 2. Ten gof this material is suspended in 100 mL sterile 5% NaOH solution andstirred gently for two hours at 5° C. (giving a pH around pH 13). Thesuspension then is diluted 1:1 in sterile, distilled water and thenfiltered through a 1 u membrane to remove undissolved particulatematerial. The pH of the filtered solution then is adjusted to 10 by theaddition of SM HCl and then dialysed against 2 L distilled water (pH 10)in a Pelicon system using a 10.000 D limiting membrane. The solutionthen can be sterilised by passage through a 0.45μ membrane and the pH ofthe solution may be adjusted as desired. The soluble glucan so producedis useful as a pharmaceutical product.

Gel permeation chromatography (Waters Styragel HR 5E® column, effectivemolecular weight range of 10×10⁴ to 4.0×10⁶ daltons) of the solubleglucan showed the material was essentially homogenous with a very narrowmolecular weight spread, having an average molecular weight of 140,000daltons and a polydispersity index of 2.564. In this determination thesolvent is DMSO and the column flow rate is 1 ml/minute.

EXAMPLE 4

A protocol for the preparation of polymerised, soluble glucan accordingto the present invention is as follows.

Microparticulate Sc-glucan is produced as detailed in Example 2. Fifteeng of this material is suspended in 100 mL sterile 5% NaOH solution andstirred gently for two hours at 5° C. The suspension then is centrifugedat 1000 g to remove undissolved particulate material. The pH of thesolution then is adjusted to 10 by the addition of 5M HCl and thendialysed against 2 L distilled water (pH 10) in a Pelicon system using a10.000 D limiting membrane. The pH then is adjusted to 7.5 by thefurther addition of hydrochloric acid producing a viscous gel which isuseful as a pharmaceutical product.

Gel permeation chromatography showed the material was essentiallyhomogenous with a very narrow molecular weight spread, having an averagemolecular weight of 320,000 daltons and a polydispersity index of 2.2.

EXAMPLE 5

A model of delayed wound healing was developed in rats to test theability of microparticulate Sc-glucan to promote wound healing indysfunctional wounds. The breaking strength of seven day-old skin woundsin inbred young adult laboratory rats is determined as outlined earlierbut the rats in this case are treated with drugs intended to depress thehealing response. This is achieved by daily treatment from the time ofwounding with a combination of prednisone (1 mg/kg), cyclosporin A (5mg/kg) and azothioprine (2 mg/kg). This triple drug therapy provides arange of depressive effects on macrophages, lymphocytes and vascularendothelium.

Table 9 summarizes the results of the use of Sc-glucan in this model.Tile effect of the triple drug therapy was to reduce significantly(p<0.01) the breaking strength of the wound at seven days. A singleapplication of 1 mg of microparticulate Sc-glucan (per 5 cm linearlength skin wound) produced by the process of the present inventionsuccessfully antagonized the depressive effect of the triple drugtherapy, returning the breaking strength of the wound to that seen innormal immunocompetent rats.

TABLE 9 Effect of topical Sc-glucan therapy on the breaking strength ofskin wounds in rats with and without drug-induced depressed woundhealing. Wound breaking Group Drug treatment Glucan treatment strength(g) (mean) 1 None None 422 2 Yes None 275 3 Yes Yes 442

EXAMPLE 6

Glucan Formulations

A topical preparations for human and veterinary applications wereprepared from the following components:

TOPICAL CREAM β-1,3-glucan (microparticulate) BP 1 mg/g Zinc stearate BP3 mg/g Cetomacrogol 1000 BP 20 mg/g Cetostearyl alcohol BP 80 mg/gPhenoxyethanol BPC 1973 5 μL/g Glycerol BP 60 mg/g Arachis oil BP 40mg/g Purified water BP to 1 g

This formulation may be referred to as Formulation #1.

A powder for topical application was prepared from the followingcomponents:

TOPICAL POWDER β-1,3-glucan (microparticulate) 100 mg/g Maize corn flourBP 900 mg/g

This formulation may be referred to as Formulation #2.

A topical cream was prepared by mixing the following components:

TOPICAL CREAM Paraffin oil 80 ml Olive oil 60 ml Anhydrous lanolincetomacrogol 1000 60 g Stearic acid Cetostearyl alcohol 58 g Glycerylmonostearate phenoxyethanol 60 g Oleic acid glycerol 25 ml Water 1200 mlTriethanolamine 27 ml Soluble glucan of Example 3 20 ml

This formulation may be referred to as Formulation #3 and provides acream containing 5 mg soluble glucan per g.

Formulations #1 to #3 were varied by incorporating glucan in the form ofa gel. These may be referred to as Formulations #1A to #3A.

EXAMPLE 7

A decubitus ulcer was treated successfully in a human patient usingFormulation #1.

The patient was a ninety year old male stroke victim who had beenhospitalized for ten years and who was essentially bed-ridden. Adecubitus ulcer had developed on the right buttock in 1986 and persisteddespite regular medical and nursing attention. By 1988 the ulcer hadgrown to a diameter of 8 cm and to a depth of 4 cm. Conventionaltreatments consisting of regular wound cleansing, application ofprotective dressings and body positioning to minimize pressure to theulcer had failed to halt the progressive deterioration of the ulcer.

Treatment with Sc-glucan was commenced and involved topical applicationusing Formulation #1. Daily topical treatments were carried out for oneweek and then ceased. Two weeks after treatment the ulcer was totallyhealed; epithelialization was complete and there was no visible scarformation.

EXAMPLE 8

A patient (Mr G W) suffering from persistent leg ulcers was treated withglucan (Formulation #1).

The patient was a fifty three year old male who suffered a sportinginjury which included a fractured ankle. Following this injury the anklewas reconstructed twice. After the second reconstruction the wound didnot heal and four venostasis ulcers developed despite the use ofsystemic and topical bactericides and antibiotics.

Following five successive daily applications of the glucan containingformulation 1 wound healing cream of Example 5 to three of the ulcers,one originally measuring 3.8 cm×1.9 cm completely healed in ten days;one measuring 10.2 cm×3.8 cm was reduced to 6.3 cm×1.3 cm during the tenday treatment period; and a further ulcer measuring 3.8 cm×1.9 cm wasreduced to 2.5 cm×1.2 cm. The treatment was recommenced on the tenth dayand after two further cycles of treatment comprising cream applicationfor seven days and no treatment for seven days the latter two ulcerscompletely healed after four weeks. Treatment of the fourth ulcer (10cm×9 cm) involving two exposed tendons and extensive tissue necrosis wascommenced shortly thereafter. After ten days of daily treatment, therewas clear evidence of epithelial regrowth and granulation tissue leadingto coverage of the exposed tendons by granulation tissue and overallreduction in wound size to 8 cm×7 cm.

The patient had never observed such positive results from any previoustreatment.

EXAMPLE 9

The posterior aspect of the forearm of a six year old thoroughbredstallion was severely traumatized in a fight with another stallioncreating a deep cavity with an external hole some 40 cm×20 cm in area.Initial treatment was irrigation with disinfectant and antibioticsolutions but after several days the severity of the injury became moreapparent and appeared to be worsening. There was extensive and deepsloughing occurring with necrosis of deep tissues including ligament andtendons and associated muscle masses—some tendon remnants were presentas unhealthy looking strands and the animal could not bear weight. Theaffected area was treated at that time by topical application ofFormulation #1 of Example 5.

There was an immediate and profound response to glucan treatment.

The sequence of the clinical response to treatment was as follows:

24 hr post-treatment: Necrosis lessened with reduction in suppuration.

36 hr post-treatment: Marked improvement in appearance of wound withtissue showing vitality.

72 hr post-treatment: Whole area filling in rapidly with ligament andtendon remnants being included in new tissue.

96 hr post-treatment: General appearance of good rapid healthy healingwith peripheral epithelialization evident.

The wound ultimately completely closed after 12 days of treatment andwith minimal scarring.

The animal at that time was weight-bearing on all legs.

EXAMPLE 10

Four adult rats (male. Wistar, inbred) had their left femurs brokenunder anaesthesia using externally-applied force. The fracture site waslocated by external palpation and a 21-gauge needle then introducedthrough the skin over the fracture site and positioned between thefractured ends of the femur. The fracture then was immobilised in thestandard way by insertion of an intra-medullary pin through the kneejoint to emerge through the femoral head. In two rats, 2 mL of colloidalglucan produced as per Example 4 were injected into the fracture sitevia the previously-positioned needle. In the other two rats, 2 mL ofsaline was injected instead of glucan.

The needle then was withdrawn and the rats allowed to recover from theanaesthetic. Twelve days later the rats were killed, the intra-medullarypins removed and the fractured femurs isolated for visualisation of thefracture site and determination of the strength of the healing response.In the two control (saline) rats, the fracture site was contained withina rudimentary callus and was able to be displaced readily by torsion ofthe upper and lower femoral shafts. In the two glucan-treated rats, thecallus was further advanced, being firmer and considerably greater forcewas required to displace the fractured ends of femur. It was concludedthat the effect of the glucan had been to accelerate callus formation,leading to a firmed bond of the fracture site at 12 days post-fracture.

EXAMPLE 11

A 50 year-old Caucasian male exposed an area of skin approximately 4cm×12 cm on the inner aspect of both forearms to direct sunlight for aperiod of 40 minutes. Both areas were exposed under identical conditionsand both forearms had similar levels of skin pigmentation. Each exposedarea was divided into 4 equal patches (4 cm×3 cm) which were delineatedby indelible ink. On each forearm, 1 gm of sun-cream (SPF 10) wasapplied to one of the end patches prior to sun-exposure; the remainingpatches were untreated at this time. Two hours following sun-exposureSc-glucan (Formulation #3 from Example 6) was applied to the secondpatches, the third patch was left untreated, and 2 gm of Formulation #3(Example 6) base without Sc-glucan applied to the fourth patch. Theorder of treatment was reversed on each forearm. The skin patches wereexamined 24 hours following sun-exposure and the degree of rednessassessed visually by scoring 0, +, ++, +++and ++++. The results were asfollows:

untreated ++++ SPF 10 + cream base only ++++ glucan + cream base ++

The glucan effected considerable reduction of skin redness. Hence,glucan ameliorated the clinical response to sun damage.

EXAMPLE 12

Albino Skh:HR-1 hairless mice were irradiated daily with U.V. light fora period of 12 weeks. After each daily irradiation, mice were paintedwith glucan cream of (Formulation #3), cream base alone or untreated.Results are shown in Table 10.

Table 10

Mean no. of pre-malignant (papillomas, hyperkeratoses,kerato-acanthomas) and malignant (carcinomas) in albino Skh:HR-1hairless mice, following 12 weeks ultraviolet irradiation painted with0.1 mld of either cream base lotion or Sc-glucan (7 mg/day) and creambase each day.

Mean no. skin tumours per mouse no. Weeks Treatment mice 11 14 17 19 21Cream base only 20 0  1.7 ± 2.7  4.7 ± 3.9 7.6 ± 3.9 13.3 ± 8.3Sc-glucan + 20 0.05 ± 0.2 0.05 ± 0.2 0.95 ± 1.9 1.7 ± 2.6  4.6 ± 4.7*cream base *p = 0.004

Mice Irradiated by UV painted with neither Sc-glucan and cream base, orcream base alone, registered as many tumours as mice painted with creambase alone (data not shown).

EXAMPLE 13

Mice are exposed daily for ten weeks to a minimal erythemal dose of U.V.light which stimulates the toxic effects of sunlight on skin. Each dailyexposure of U.V. light induces a mild erythema and oedema lasting up to25 hours which mimmics a mild ‘sun-burn’ in humans. Mice were eithertreated with Formulation #3 after U.V. light exposure (group 1) ortreated with base lotion containing no glucan (group 2). At six weeksnotable skin thickening (and consequential wrinkling) was observed forgroup 2 mice. Mice of group 1 were largely protected from these effects.Erythema was not observed in group 1 mice over the treatment period.FIG. 1 depicts the results obtained in one test. After 6 weeks, glucantreated mice (-□-) showed appreciably less skin fold thickness thanuntreated mice (-⋄-)

REFERENCES

Bacon J S D, Farmer, V C, Jones D, Taylor I F, “The glucan component ofthe cell wall of baker's yeast (Saccharomyces cerevisiae) considered inrelation to its ultrastructure”, Biochem. J., 114, 557-567 (1969)

Bak B., Jensen K S, “Standardization of Tibial Fractures in the Rat”Bone. 13,289-295 (1992)

Canfield P J, Greenoak G E, Reeve V E, Gallagher C H, “Characterisationof UV induced keratoancanthoma-like lesions in HRA/Skh-1 mice and theircomparison with keratoacanthomas in man”, Pathology, 17(4), 613-616(1985)

Cook J A, Holbrook T W, Parker B W, “Visceral leishmaniasis in mice:protective effect of glucan”, Journal of the ReticuloendothelialSociety, 27, 567-573 (1980)

Czop J K, Austen K F, “Generation of leukotrienes by human monocytesupon stimulation of their β-glucan receptor during phagocytosis”,Proceedings of the National Academy of Sciences (USA), 82, 2751-2755(1985)

Di Luzio N R, Williams D L, McNamee R B, Edwards B F, Kitahama A,“Comparative tumor-inhibitory and antibacterial activity of soluble andparticulate glucan”, Int J Cancer. 24, 773-779 (1979)

Deimann, Fahimi, Journal of Experimental Medicine, 149, 883-897 (1979)

Hassid W Z, Joslyn M A, McCready R M, “The molecular constitution of aninsoluble polysaccharide from yeast, Saccharomyces cerevisiae”, Journalof the American Chemical Society, 63, 295-298 (1941)

Kelly G E, Lui. W, “Accelerated wound healing in normal andimmunosuppressed animals ”, Norvet Research Pty Ltd, 1994, ReportG94003.

Maeda Y Y, Chihara G, “The effects of neonatal thymectomy on theantitumour activity of lentinan, carboxymethylpachymaran and zymosan,and their effects on various immune responses”, International Journal ofCancer, 11, 153-161 (1973)

Manners D J, Masson A J, Patterson J C, “The structure of aβ-(1,3)-D-glucan from yeast cell walls” Biochem J, 135, 19-30 (1973)

Mansell P W A, Ichinose H, Reed R J, Krementz E T, McNamee R, Di Luzio NR, “Macrophage-mediated destruction of human malignant cells in viio”,Journal of the National Cancer Institute, 54. 571-580 (1975)

Niskanen, Cancer Research, 38, 1406-1409 (1978)

Patchen, Lotzova, Experimental Haematology 8, 409422 (1980)

Riggi S, Di Luzio N R, “Identification of a RE stimulating agent inzymosan”, American Journal of Physiology 200, 297-300 (1961)

Sherwood E R, Williams D L, Di Luzio N R, “Glucan stimulates productionof antitumor cytolytic/cytostatic factor(s) by macrophages”, Journal ofBiological Response Modifiers, 5, 504-526 (1986)

Sherwood E R, Williams D L, McNamee R B, Jones E L, Browder I W, DiLuzio N R, “Enhancement of interleukin-1 and interleukin-2 production bysoluble glucan”, International Journal of Immunopharmacology, 9, 261-267(1987)

Williams D L, Pretus H A, McNamee R B, Jones E L, Ensley H E, Browder IW, Di Luzio N R, “Development, physicochemical characterization andpreclinical efficacy evaluation of a water soluble glucan sulfatederived from Saccharomyces cerevisiae” Immunopharmacol, 22, 139-156(1991)

Williams D L, Cook J A, Hoffmann E O, Di Luzio N R, “Protective effectof glucan in experimentally induced candidiasis”, Journal of theReticuloendothelial Society 23, 479490 (1978)

Williams D L, Browder I W, Di Luzio N R, “Immunotherapeutic modificationof E. coli-induced experimental peritonitis and bacteremia by glucan”,Surgery, 93, 448454 (1983)

Williams D L, Sherwood E R, McNamee R B, Jones E L, Di Luzio N R,“Therapeutic efficacy of glucan in a murine model of hepatic metastaticdisease”, Hepatology 5, 198-206 (1985)

Williams D L, McNamee R B, Jones E L, Pretus H A, Ensley H E, Browder IW, Di Luzio N R “A method for the solubilization of a (1-3)-β-D-glucanisolated from Saccharomyces cerevisiae”, Carbohydrate Research, 219,203-213 (1991)

The references referred to herein are incorporated by reference.

What is claimed is:
 1. A process for isolation of β-(1,3)(1,6)-glucanfrom a glucan containing cellular source which comprises the steps of:(a) extracting glucan containing cells with alkali and heat, in order toremove alkali soluble components; (b) acid extracting the cells of step(a) with an acid and heat to form a suspension; (c) separating branchedβ-(1,3)(1,6) glucan from unbranched β-(1,3) glucan by (i) extracting thesuspension obtained of step (b) or recovered hydrolyzed cells with anorganic solvent which is non-miscible with water and which has a densitygreater than that of water to yield; an aqueous phase containingbranched β-(1,3)(1,6)-glucan particulate material and essentially freeof unbranched β-(1,3) glucan, an interface phase containing unbranchedβ-(1,3) glucan and residual non-glucan contaminants, and an organicsolvent containing phase, and (ii) separating the resultant aqueousphase from the interface phase and the organic solvent containing phase;and (d) drying the branched particulate β-(1,3)(1,6) glucan material inthe separated aqueous phase to give microparticulate branchedβ-(1,3)(1,6) glucan.
 2. A process according to claim 1, which is aprocess for isolating soluble branched β-(1,3)(1,6) glucan, wherein step(d) is omitted and the pH of the separated aqueous phase is raised so asto effect solubilization of the branched particulate β-(1,3)(1,6) glucanat a temperature of less than about 60° C.
 3. A process according toclaim 2, wherein the step of raising the pH of the separated aqueousphase is conducted at a temperature of between about 2° C. and about 8°C. and wherein the soluble glucan has a polydispersity index suitablefor use as a pharmaceutical product.
 4. A process according to claim 2wherein the pH of the separated aqueous phase after solubilization ofthe branched particulate β-(1,3)(1,6) glucan is adjusted to be betweenabout pH 9 to about pH 10, and the resultant soluble branchedβ-(1,3)(1,6) glucan is admixed with one or more pharmaceuticallyacceptable carriers or excipients.
 5. A process according to claim 2wherein the pH of the separated aqueous phase after solubilization ofthe branched particulate β-(1,3)(1,6) glucan is adjusted from about 7 toabout 8 so as to form a gel which optionally is admixed with one or morepharmaceutically acceptable carriers or excipients.
 6. A processaccording to claim 1, which is a process for isolating soluble branchedβ-(1,3)(1,6) glucan, wherein the microparticulate branched β-(1,3)(1,6)glucan of step (d) is suspended in an aqueous alkali solution so as toeffect solubilization of the branched β-(1,3)(1,6) particulate glucan ata temperature of less than about 60° C.
 7. A process according to claim1 wherein the acid used at step (b) is selected from acetic acid, formicacid, phosphoric acid, and hydrochloric acid.
 8. A process according toclaim 1 wherein the pH of the acid of step (b) is from about 2 to about6.
 9. A process according to claim 1 wherein the organic solvent of step(c) is selected from chloroform, methylchloroform, dichloromethane,tetrachloroethane, and carbon tetrachloride.
 10. A process according toclaim 11 wherein said organic solvent of step (c) is chloroform.
 11. Theprocess of claim 1 wherein said branched β-(1,3)(1,6)-glucan particulatematerial contains 0.3% or less protein by weight.
 12. A method oftreatment whereby glucan, prepared according to claim 1 and optionallyin association with one or more pharmaceutically, veterinarily oragriculturally acceptable carriers or excipients, is administered to asubject suffering from bone fracture, ulcers caused by physical trauma,impaired blood flow, infection, or neoplasia, or is administered to asubject in need of the enhancement of fixation of implanted orthopaedicdevices.
 13. A process for separating branched β-(13)(1,6) glucan fromunbranched β-(1,3) glucan in an aqueous acidic suspension containingbranched β-(1,3)(1,6) glucan, unbranched β-(1,3) glucan, and residualnon-glucan contaminants, the process comprising the steps of: (a)extracting the suspension with an organic solvent to yield an aqueousphase containing branched β-(1,3)(1,6) glucan particulate material andessentially free of unbranched β-(1,3) glucan, and interface phasecontaining unbranched β-(1,3) glucan and residual non-glucancontaminants, and an organic solvent containing phase, wherein theorganic solvent is non-miscible in water and has a density greater thanthat of water; and (b) separating the resultant aqueous phase from, theinterface phase and the organic solvent containing phase; and (c)solubilizing the branched β-(1,3)(1,6) glucan particulate material inthe separated aqueous phase by increasing the pH of the separatedaqueous phase at a temperature of less than 60° C.
 14. The process ofclaim 13 wherein the temperature of the solubilization step is betweenabout 2° C. and about 8° C.
 15. The process according to claim 13wherein the pH of the aqueous acidic suspension is from about 2 to about6.
 16. The process according to claim 13 wherein the organic solvent isselected from the group consisting of chloroform, methylchloroform,dichloromethane, tetrachloroethane and carbon tetrachloride.
 17. Aprocess according to claim 16 wherein said solvent is chloroform.
 18. Aprocess for separating branched β-(1,3)(1,6) glucan from unbranchedβ-(1,3) glucan in an aqueous acidic suspension containing branchedβ-(1,3)(1,6) glucan, unbranched β-(1,3) glucan, and residual non-glucancontaminants, the process comprising the steps of: (a) extracting thesuspension with an organic solvent to yield an aqueous phase containingbranched β-(1,3)(1,6) glucan particulate material and essentially freeof unbranched β-(1,3) glucan, an interface phase containing unbranchedβ-(1,3) glucan and residual non-glucan contaminants, and an organicsolvent containing organic solvent is non-miscible in water and has adensity greater than that of water; (b) separating the resultant aqueousphase from the interface phase and the organic solvent containing phase;(c) drying the branched β-(1,3)(1,6) glucan particulate material in theseparated aqueous phase to give microparticulate branched β-(1,3)(1,6)glucan; and (d) solubilizing the microparticulate branched β-(1,3)(1,6)glucan by suspending the microparticulate branched β-(1,3)(1,6) glucanin an aqueous alkali solution at a temperature of less than 60°.
 19. Theprocess of claim 18 wherein the temperature of the solubilization stepis between about 2° C. and about 8° C.
 20. A process for separatingbranched β-(1,3)(1,6) glucan from unbranched β-(1,3) glucan in anaqueous acidic suspension containing branched β-(1,3)(1,6) glucan,unbranched β-(1,3) glucan, and residual non-glucan contaminants, theprocess comprising the steps of: (a) extracting the suspension with anorganic solvent to yield an aqueous phase containing branchedβ-(1,3)(1,6) glucan particulate material and essentially free ofunbranched β-(1,3) glucan, an interface phase containing unbranchedβ-(1,3) glucan and residual non-glucan contaminants, and an organicsolvent containing phase, wherein the organic solvent is non-miscible inwater and has a density greater than that of water; and (b) separatingthe resultant aqueous phase from the interface phase and the organicsolvent containing phase; (c) drying the branched β-(1,3)(1,6) glucanparticulate material in the separated aqueous phase to givemicroparticulate branched β-(1,3)(1,6) glucan; and (d) solubilizing themicroparticulate branched β-(1,3)(1,6) glucan by suspending themicroparticulate branched α-(1,3)(1,6) glucan in an aqueous alkalisolution at a temperature of less than 60° C. to yield soluble branchedβ-(1,3)(1,6) glucan having a polydispersity index of about 5 or less.21. The process of claim 20 wherein the temperature of thesolubilization step is between about 2° C. and about 8° C.
 22. Theprocess of claim 20 wherein said polydispersity index is about 3 orless.